The immune system eliminates malignant cells by recognizing them as foreign and then clearing them from the body. To accomplish this, the immune system invokes both an antibody response and a cellular response. Both these responses require interaction among a number of different cells of the immune system (Abbas, Cellular and Molecular Immunology, 2000).
An immune reaction typically begins with a T lymphocyte (T cell) that has on its surface a T cell receptor (TCR) that binds to an antigen derived peptide associated with a class II major histo-compatibility complex (MHC) molecule. The T cell also expresses on its surface various polypeptides, which are referred to as “ligands” because they bind to receptors on cells associated with an immune-mediated response, as described in more detail below. When the T cell receptor binds to a MHC-associated antigen, such as antigen derived from a malignant cell, it becomes activated and expresses a ligand on its surface. The ligand is only present on the cell surface for a short time, and once it has been removed from the surface of the cell, the T cell's ability to bind a receptor-bearing cell is lost. One such ligand is called CD154.
CD154 is one member of a larger family of ligands, collectively referred to as the TNF superfamily (Gruss et al, Cytokines Mol Ther, 1:75-105, 1995 and Locksley et al, Cell, 104:487-501, 2001). Members of the TNF superfamily include Fas ligand (“FasL”), TNFα, LTα, lymphotoxin (TNFβ), CD154, TRAIL, CD70, CD30 ligand, 4-1BB ligand, APRIL, TWEAK, RANK ligand, LIGHT, AITR ligand, ectodysplasin, BLYS, VEGI, and OX40 ligand. TNF superfamily members share a conserved secondary structure comprising four domains: domain I, the intracellular domain; domain II, which spans the cell membrane and is known as the transmembrane domain; domain III, which consists of the extracellular amino acids closest to the cell membrane; and domain IV, the distal extracellular domain (Kipps et al., WO98/26061 published Jun. 18, 1998). Typically, at least a part of domain IV can be cleaved from the parent molecule. The cleaved fragment often exhibits the same biological activity of the intact ligand and is conventionally referred to as a “soluble form” of the TNF family member.
I) Biological Activity of CD154
The interactions between CD154 (also known as CD40 ligand) and its cognate receptor, CD40, are critical for immune recognition. (Banchereau J. et al., Annu. Rev. Immunol. 12:881-922, 1994; Laman J. D. et al., Crit. Rev. Immunol., 16:59-108, 1996). CD154 is transiently expressed on CD4+ T cells following T cell receptor engagement by antigen presenting cells through MHC class II molecules. (Roy M. et al., J. Immunol., 151:2497-2510, 1993; Hepmann P. et al., Eur. J. Immunol., 23:961-964, 1993; Castle B. E. et al., J. Immunol., 151:1777-1788, 1993; Cantwell M. et al., Nat. Med., 3:984-989, 1997). This, in turn, can cause activation of CD40-expressing antigen presenting cells (APCs), including B cells, dendritic cells, monocytes, and macrophages. (Ranheim E. A. et al., J. Exp. Med., 177:925-935, 1993; Ranheim E. A. et al., Cell. Immunol., 161:226-235, 1995). Such CD40 activated cells can set off a cascade of immune-activating events that lead to a specific and effective immune response against foreign antigens, such as viruses or tumors. The importance of interactions between CD40 and CD154 is underscored by the finding that individuals who have inherited defects in the ligand for CD40 have profound immune deficiency. (Korthauer J. et al., Nature, 361:539-541, 1993; Aruffo A. et al., Cell., 72:291-300, 1993). Such patients have an immune deficiency syndrome associated with impaired germinal center formation, defective isotype switching, and marked susceptibility to various bacterial and viral pathogens.
Because CD154 is such a critical molecule in immune regulation, several mechanisms control human CD154 expression. First, membrane-expressed CD154 can be cleaved and an extracellular portion of CD154 capable of binding the CD154 receptor, CD40, is released as a soluble molecule. Proteolytic cleavage enzymes have been shown to cleave human CD154 at different sites along the ligand, and release a soluble form of CD154 that is capable of binding to CD40 and stimulating an immune response. (Pietravalle F. et al., J. Biol. Chem., 271:5965-5967, 1996; Pietravalle F. et al., Eur. J. Immunol., 26:725-728, 1996). For instance, one study has shown that CD154 is cleaved between Phe 111 and Ala 123 (Pietravalle F. et al., Eur. J. Immunol., 26:725-728, 1996), and cleavage has also been reported at Met 113. Second, CD154 interaction with its cognate receptor can induce rapid downmodulation of CD154 surface expression. (Cantwell M. et al., Nat. Med., 3:984-989, 1997). Third, CD154 gene transcription is tightly regulated with maximum ligand expression 4 to 6 hours after TCR ligation followed by rapid decreases in CD154 RNA and protein synthesis. (Id.) Together, these regulatory mechanisms ensure specificity of an immune response to a specific antigen. The importance of maintaining tight control of CD154 expression is illustrated in individuals with systemic lupus erythematosus (SLE). These patients appear to hyper-express CD154 as well as possess elevated levels of soluble CD154 in their plasma, suggesting uncontrolled CD154 expression contributes to SLE disease activity. (Kato K. et al., J. Clin. Invest., 101:1133-1141, 1998; Vakkalanka R. K., Arthritis Rheum., 42:871-881, 1999).
The potential for using CD154 for immunotherapy is under active investigation. Because CD154 is a potent immune activator, CD154 as a cancer therapy is a main focus of research because neoplastic cells are generally poor presenters of antigen and unable to stimulate vigorous anti-tumor responses. For example, chronic lymphocytic leukemia (CLL) B cells modified to express CD154 using a replication defective adenovirus vector can enhance CLL antigen presentation and induce autologous T cell cytotoxicity towards nonmodified CLL B cells. (Kato K. et al., J. Clin. Invest., 101:1133-1141, 1998). Moreover, a phase-I clinical study using Ad-CD154 modified CLL B cells showed promising therapeutic results. (Wierda W. G. et al., Blood, 96:2917-2924, 2000). Similarly, other studies showed that modification of a range of tumor types to express CD154 can induce effective anti-tumor immune responses in animal models.
Studies manipulating B cells and other tumors work by either enhancing the antigen presentation of the neoplastic cell itself, as is the case for CLL and B cell lymphoma, or by activating bystander antigen presenting cells, such as dendritic cells that can initiate an anti-tumor immune response, as is the case for CD40-negative tumors. However, additional studies also suggest CD154 might have a direct growth-inhibitory effect on certain tumors, especially carcinomas of the breast. (Tong A. W. et al., Clin. Cancer Des., 7:691-703, 2001; Hirano A., Blood, 93:2999-3007, 1999). In addition, there is evidence that growth of some types of lymphoma can be directly inhibited by CD40 ligation. (Wilsey J. A. et al., J. Immunol., 158:2932-2938, 1997). As such, a wide range of tumors should be amenable to CD154 immunotherapy.
II) Drawbacks of Current CD154 Constructs
Although CD154 is a potentially powerful therapeutic, the form of CD154 used in clinical therapies will likely have a major impact on both safety and efficacy.
For example, recombinant soluble CD154 (rsCD154) composed only of the extracellular, receptor-binding domain of CD154 is functional. (Armitage R. J., Eur. J. Immunol., 23:2326-2331, 1993; Lane P., J. Exp. Med., 177:1209-1213, 1993). However rsCD154 is not as effective as native CD154 expressed on the cell membrane to induce CD40 signaling because optimal signaling requires multimerization of the CD40 receptors at the cell surface. (Schwabe R. F. et al., Hybridoma, 16:217-226, 1997). As a result, ligand-multimerization domains have been engineered, such as leucine zippers or CD8 domains, onto the n-terminal domain of rsCD154 to enhance receptor signaling. (Lans P., et al., J. Exp. Med. 177:1209-1213, 1993; Morris A. E., J. Biol. Chem. 274:418-423, 1999). Likewise, soluble CD154 is not optimal for cross-linking CD40 since it does not provide as strong a stimulation of antigen-presenting cells compared to membrane-expressed CD154.
In addition, soluble reagents that mediate CD40 signaling can trigger adverse physiological effects. For example, mice injected with soluble CD154-CD8 fusion protein developed pulmonary inflammation. (Wiley J. A. et al., J. Immunol., 158:2932-2938, 1997). Likewise, administration of CD40-activating monoclonal antibody to immunocompromised mice induced intestinal lesions that were fatal. (Hixon J. A. et al., Biol. Blood Marrow Transplant., 7:136-143, 2001) The toxicity associated with systemic administration of soluble CD154 appears to be a general feature of the TNF family since adverse effects are also seen following administration of soluble TNF-α, FasL, and TRAIL.
Another drawback of soluble CD154 is the short half-life of soluble TNF family members following systemic administration. (Spriss D. R. et al., Ciba Found. Symp., 131:206-227, 1987; Funahashi I. et al., Br. J. Cancer, 67:447-455). This short half-life would require delivery of either higher doses of rsCD154 or continuous infusion over time, which not only increases the chances of toxicity but also would require isolation of large amounts of rsCD154 protein, a difficult and time-consuming process.
Due to the inherent problems using soluble CD154, membrane-expressed full-length human CD154 seems the better alternative. However, native human CD154 also possesses characteristics that might limit its efficacy or safety. As previously mentioned, full-length CD154 is cleaved and released as a soluble molecule, potentially allowing for similar toxicities described for rsCD154. In addition, proteolytic cleavage of membrane-bound CD154 might decrease its functional activity. Although deletion of putative cleavage sites from CD154 can decrease its metabolism, this does not completely eliminate CD154 processing since multiple proteolytic cleavage sites exist. (Mazzei G. J. et al., J. Biol. Chem., 270:7025-7028, 1995; Pistravalle F. et al., J. Biol. Chem., 271:5965-5967, 1996). Moreover, a less apparent problem associated with using full-length human CD154 is its cell-type specific expression. For example, certain cell types, especially cells of B-cell origin, preclude expression of human CD154. (Kato K. et al., J. Clin. Invest., 101:1133-1141, 1998; Cantwell M. et al., Nat. Med., 3:984-989, 1997).
Interestingly, murine CD154 (mCD154) appears more advantageous than either native human CD154 or rsCD154 for therapeutic uses. Murine CD154 is relatively resistant to proteolytic cleavage in comparison to human CD154. Moreover, mCD154 is expressed by most cell types, including cells of B-cell origin that preclude human CD154 expression, often referred to as CD40+ cells. (Id.) As such, mCD154 was expressed in the clinical trial of CD154 gene therapy of one type of CD40+ cell, a CLL cell. (Wierda W. G., Blood, 96:2917-2924 (2000).
Still, mCD154 use in humans presents its own problems. For example, following repeated injections of Ad-CD154 modified CLL cells to patients, the reduction in leukemic cells decreased with each subsequent injection. Three of four CLL patients became refractory to the activity of mCD154-expressing cells by the fifth repeat injection. This loss of activity is likely due to the development of antibodies against the murine CD154 molecule making further treatments impossible. Assays to determine the formation of binding and neutralizing antibodies against CD154 showed anti-murine CD154 antibodies developed by the third repeat injection of Ad-mCD154 transduced CLL cells. In addition, the anti-CD154 antibodies could also neutralize murine CD154 function. Thus, despite the overall safety, expression stability, and short-term efficacy of mCD154, long-term repeated administration of mCD154 in humans will be difficult.
Given the disadvantages of current CD154 constructs, there is clearly a need for a preferred CD154 construct for disease therapy that possesses properties found in both human CD154 and murine CD154. A preferred CD154 construct would be expressed on diverse cell types, including lymphoid cells of B-cell origin. In addition, the CD154 construct would be membrane-stabilized and resistant to proteolytic cleavage, and thereby less likely to generate the soluble form of CD154. However, the preferred CD154 construct would maintain the receptor-binding function of native CD154. Both these properties are found in mCD154. Moreover, a preferred CD154 construct would not be immunogenic at the domain critical for receptor binding following administration in humans, thus avoiding functional neutralization. The present invention provides for such a CD154 construct.